Depolarization-secretion coupling is thought to occur via electrical activity leading to the entry of calcium and the subsequent secretion of transmitters. The molecular details of how calcium and other ionic currents control the release of neuropeptides from nerve terminals in the intact central nervous system (CNS), however, remain undetermined. Vasopressin (AVP) and oxytocin (OT) are synthesized by magnocellular neurons of the hypothalamus and secreted from neurohypophysial (NH) terminals; together they comprise the Hypothalamic-Neurohypophysial System (HNS). OT neurons are characterized by a high frequency discharge during suckling which leads to the pulsatile release of OT. AVP neurons are characterized by their asynchronous phasic activity (bursting) during maintained AVP release. In both cases, it is the clustering of spikes which facilitates neuropeptide release. We have discovered that there are different calcium-channel subtypes in AVP vs. OT terminals, but that their biophysical properties alone cannot explain the differential facilitation of release by such burst patterns. Therefore, we hypothesize that autocrine/paracrine feedback effects might help determine the efficacy of different bursting patterns of electrical activity to facilitate release of AVP vs. OT. ATP is co-released with the HNS peptides. Purines, such as ATP and adenosine, interact with specific receptors on neurons, leading to a variety of effects. It is not known; however, at what specific receptors these effects occur at synapses in the CNS. We have characterized the electrical and secretory effects on the HNS terminals by purines via P2X2, P2X3 and A1 receptors, including effects on ionic conductances in these CNS terminals. The HNS affords the unique opportunity of unraveling the complicated effects of endogenous purines in the CNS by comparing such effects on isolated terminals vs. on the intact, whole system. The goal of the research proposed here is to determine membrane mechanisms that mediate endogenous purinergic-induced efficacy of neuropeptide secretion during physiological patterns of electrical stimulation. To achieve these objectives, perforated and loose patch-clamp recordings of resting potential, calcium- and action potential-currents will be made from identified, isolated nerve terminals vs. intact preparations of the HNS of adult rats and mice. Effects on release will be compared between the intact HNS and isolated NH terminals by the use of ELISAs and capacitance measurements. This proposal now takes advantages of newly available genetic tools that facilitate the elucidation of the function of these purinergic receptors with greater specificity than is possible with traditional antagonist drugs. Furthermore, since all synaptic vesicles/neurosecretory granules appear to contain ATP, these purinergic feedback mechanisms could be physiologically important at many other synapses in the CNS. These receptor knockout studies will provide a unique opportunity to determine if endogenous purinergic feedback regulation occurs at the terminals of CNS neurons. PUBLIC HEALTH RELEVANCE: The purines, ATP and Adenosine, mediate effects that include nociception and mechanosensory transduction, depression of neurotransmission, sleep induction, anti-ischemia, ethanol-induced motor incoordination, autonomic control of cardiac function, and renal sodium retention. The use of pharmacological inhibitors has suggested a key role for purinergic receptors in synaptic plasticity and possible roles in Parkinson's and Huntington's diseases; however, these antagonists are not specific enough for individual members of the purinergic receptor family. This proposal takes advantages of newly available genetic tools (knockouts) that facilitate the elucidation of the function of these purinergic receptors with greater specificity than is generally possible with traditional antagonist drugs and gives hope for the determination of such feedback effects at CNS synapses. Only then can therapeutic drugs be targeted to physiologically relevant purinergic receptors to alleviate such diseases.